ABSTRACT
Plasmids are a foundational tool for basic and applied research across all subfields of biology. Increasingly, researchers in synthetic biology are relying on and developing massive libraries of plasmids as vectors for directed evolution, combinatorial gene circuit tests, and for CRISPR multiplexing. Verification of plasmid sequences following synthesis is a crucial quality control step that creates a bottleneck in plasmid fabrication workflows. Crucially, researchers often elect to forego the cumbersome verification step, potentially leading to reproducibility and-depending on the application-security issues. In order to facilitate plasmid verification to improve the quality and reproducibility of life science research, we developed a fast, simple, and open source pipeline for assembly and verification of plasmid sequences from Illumina reads. We demonstrate that our pipeline, which relies on de novo assembly, can also be used to detect contaminating sequences in plasmid samples. In addition to presenting our pipeline, we discuss the role for verification and quality control in the increasingly complex life science workflows ushered in by synthetic biology.
Subject(s)
Databases, Genetic , Plasmids/genetics , Sequence Analysis, DNA/methods , Base Composition , Base Sequence , Escherichia coli/geneticsABSTRACT
The field of synthetic biology relies on an ever-growing supply chain of synthetic genetic material. Technologies to secure the exchange of this material are still in their infancy. Solutions proposed thus far have focused on watermarks, a dated security approach that can be used to claim authorship, but is subject to counterfeit, and does not provide any information about the integrity of the genetic material itself. In this manuscript, we describe how data encryption and digital signature algorithms can be used to ensure the integrity and authenticity of synthetic genetic constructs. Using a pilot software that generates digital signatures and other encrypted data for plasmids, we demonstrate that we can predictably extract information about the author, the identity, the integrity of plasmid sequences, and even annotations from sequencing data alone without a reference sequence, all without compromising the function of the plasmids. Encoding a digital signature into a DNA molecule provides an avenue for genetic designers to claim authorship of DNA molecules. This technology could help compliance with material transfer agreements and other licensing agreements.
Subject(s)
Algorithms , Computer Security , Software , Base Sequence , DNA/genetics , Escherichia coli/genetics , Feasibility Studies , Genetic Engineering/methods , Humans , Mutation , Pilot Projects , Plasmids/genetics , Polymorphism, Single Nucleotide , Synthetic Biology/methodsABSTRACT
Over the last 30 years, computational biologists have developed increasingly realistic mathematical models of the regulatory networks controlling the division of eukaryotic cells. These models capture data resulting from two complementary experimental approaches: low-throughput experiments aimed at extensively characterizing the functions of small numbers of genes, and large-scale genetic interaction screens that provide a systems-level perspective on the cell division process. The former is insufficient to capture the interconnectivity of the genetic control network, while the latter is fraught with irreproducibility issues. Here, we describe a hybrid approach in which the 630 genetic interactions between 36 cell-cycle genes are quantitatively estimated by high-throughput phenotyping with an unprecedented number of biological replicates. Using this approach, we identify a subset of high-confidence genetic interactions, which we use to refine a previously published mathematical model of the cell cycle. We also present a quantitative dataset of the growth rate of these mutants under six different media conditions in order to inform future cell cycle models.
Subject(s)
Cell Cycle/genetics , Saccharomyces cerevisiae/genetics , Cell Division/genetics , Computational Biology/methods , Epistasis, Genetic/genetics , Gene Expression Regulation, Fungal/genetics , Gene Regulatory Networks/genetics , High-Throughput Screening Assays/methods , Models, Theoretical , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Laboratory strains, cell lines, and other genetic materials change hands frequently in the life sciences. Despite evidence that such materials are subject to mix-ups, contamination, and accumulation of secondary mutations, verification of strains and samples is not an established part of many experimental workflows. With the plummeting cost of next generation technologies, it is conceivable that whole genome sequencing (WGS) could be applied to routine strain and sample verification in the future. To demonstrate the need for strain validation by WGS, we sequenced haploid yeast segregants derived from a popular commercial mutant collection and identified several unexpected mutations. We determined that available bioinformatics tools may be ill-suited for verification and highlight the importance of finishing reference genomes for commonly used laboratory strains.
Subject(s)
Quality Control , Whole Genome Sequencing , Biomedical Research , Genome/genetics , Mutation/genetics , Polymorphism, Single Nucleotide/genetics , Reproducibility of Results , Saccharomyces cerevisiae/genetics , Whole Genome Sequencing/methodsABSTRACT
Certain introns significantly increase mRNA accumulation by a poorly understood mechanism. These introns have no effect when located upstream, or more than ~1 Kb downstream, of the start of transcription. We tested the ability of a formerly non-stimulating intron containing 11 copies of the sequence TTNGATYTG, which is over-represented in promoter-proximal introns in Arabidopsis thaliana, to affect expression from various positions. The activity profile of this intron at different locations was similar to that of a natural intron from the UBQ10 gene, suggesting that the motif increases mRNA accumulation by the same mechanism. A series of introns with different numbers of this motif revealed that the effect on expression is linearly dependent on motif copy number up to at least 20, with each copy adding another 1.5-fold increase in mRNA accumulation. Furthermore, 6 copies of the motif stimulated mRNA accumulation to a similar degree from within an intron or when introduced into the 5'-UTR and coding sequences of an intronless construct, demonstrating that splicing is not required for this sequence to boost expression. The ability of this motif to substantially elevate expression from several hundred nucleotides downstream of the transcription start site reveals a novel type of eukaryotic gene regulation.
Subject(s)
Arabidopsis/genetics , Gene Expression Regulation, Plant/genetics , RNA Splicing/genetics , 5' Untranslated Regions/genetics , Introns/genetics , Plants, Genetically Modified/genetics , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , Transcription Initiation Site/physiology , Transcription, Genetic/geneticsABSTRACT
The rapid pace of life sciences innovations and a growing list of nontraditional actors engaging in biological research make it challenging to develop appropriate policies to protect sensitive infrastructures. To address this challenge, we developed a five-day awareness program for security professionals, including laboratory work, site visits, and lectures.
Subject(s)
Security Measures/statistics & numerical data , Synthetic Biology/standards , Biological Science Disciplines/standards , Bioterrorism/prevention & control , HumansABSTRACT
The ease of performing both forward and reverse genetics in Saccharomyces cerevisiae, along with its stable haploid state and short generation times, has made this budding yeast the consummate model eukaryote for genetics. The major advantage of using budding yeast for reverse genetics is this organism's highly efficient homology-directed repair, allowing for precise genome editing simply by introducing DNA with homology to the chromosomal target. Although plasmid- and PCR-based genome editing tools are quite efficient, they depend on rare spontaneous DNA breaks near the target sequence. Consequently, they can generate only one genomic edit at a time, and the edit must be associated with a selectable marker. However, CRISPR/Cas technology is efficient enough to permit markerless and multiplexed edits in a single step. These features have made CRISPR/Cas popular for yeast strain engineering in synthetic biology and metabolic engineering applications, but it has not been widely employed for genetic screens. In this review, we critically examine different methods to generate multi-mutant strains in systematic genetic interaction screens and discuss the potential of CRISPR/Cas to supplement or improve on these methods.
Subject(s)
CRISPR-Cas Systems , Genome, Fungal , Genomics/methods , Yeasts/genetics , Diploidy , Gene Editing , Gene Library , Genetic Engineering/methods , Genetic Testing/methods , Mutation , Saccharomyces cerevisiae/genetics , Synthetic Lethal MutationsABSTRACT
New innovation ecosystems are emerging that challenge the complex intellectual property and regulatory landscape surrounding drug development in the United States (US). A prime example is an initiative known as the Open Insulin Project. The goal of the project is to sidestep patents and enable generic manufacturers to produce cheaper insulin. However, the US regulatory environment, not patent exclusivity, is the main barrier to insulin affordability. If the Open Insulin Project succeeds in releasing an open protocol for insulin manufacturing, follow-on work could enable a number of new insulin production ecosystems, including 'home-brewed' insulin. Regulators will need to consider how to proceed in a future where commercial pharmaceuticals remain unaffordable, but patients are empowered to produce drugs for their personal use.
Subject(s)
Biomedical Research/organization & administration , Biotechnology/methods , Drugs, Generic/economics , Insulin/economics , Intellectual Property , Technology, Pharmaceutical/methods , Drugs, Generic/isolation & purification , Humans , Insulin/isolation & purification , United StatesABSTRACT
The cyber-physical nature of biotechnology raises unprecedented security concerns. Computers can be compromised by encoding malware in DNA sequences, and biological threats can be synthesized using publicly available data. Trust within the biotechnology community creates vulnerabilities at the interface between cyberspace and biology. Awareness is a prerequisite to managing these risks.
Subject(s)
Biotechnology/methods , Bioterrorism/prevention & control , Computer Security , Information Dissemination , Internet , AwarenessABSTRACT
To more precisely define the positions from which certain intronic regulatory sequences increase mRNA accumulation, the effect of a UBIQUITIN intron on gene expression was tested from six different positions surrounding the transcription start site (TSS) of a reporter gene fusion in Arabidopsis thaliana The intron increased expression from all transcribed positions but had no effect when upstream of the 5'-most TSS. While this implies that the intron must be transcribed to increase expression, the TSS changed when the intron was located in the 5'-untranslated region (UTR), suggesting that the intron affects transcription initiation. Remarkably, deleting 303 nucleotides of the promoter including all known TSSs and all but 18 nucleotides of the 5'-UTR had virtually no effect on the level of gene expression as long as an intron containing stimulatory sequences was included. Instead, transcription was initiated in normally untranscribed sequences the same distance upstream of the intron as when the promoter was intact. These results suggest that certain intronic DNA sequences play unexpectedly large roles in directing transcription initiation and constitute a previously unrecognized type of downstream regulatory element for genes transcribed by RNA polymerase II.
Subject(s)
DNA, Plant/genetics , Introns/genetics , Promoter Regions, Genetic/genetics , 5' Untranslated Regions/genetics , Arabidopsis/genetics , Arabidopsis Proteins/genetics , RNA Polymerase II/genetics , Transcription Initiation Site/physiology , Transcription, Genetic/geneticsABSTRACT
Within two years of their discovery in 1977, introns were found to have a positive effect on gene expression. Numerous examples of stimulatory introns have been described since then in very diverse organisms, including plants. In some cases, the mechanism through which the intron affects expression is readily understood. However, many introns that affect expression increase mRNA accumulation through an unknown mechanism, referred to as intron-mediated enhancement (IME). Despite several decades of research into IME, and the clear benefits of using introns to increase transgene expression, little progress has been made in understanding the mechanism of IME. Several fundamental questions regarding the role of transcription and splicing, the sequences responsible for IME, the involvement of other factors, and the relationship between introns and promoters remain unanswered. The more we learn about the properties of stimulating introns, the clearer it becomes that the effects of introns are unfamiliar and difficult to reconcile with conventional views of how transcription is controlled. We hypothesize that introns increase transcript initiation upstream of themselves by creating a localized region of accessible chromatin. Introns might represent a novel kind of downstream regulatory element for genes transcribed by RNA polymerase II.